This invention relates to vaccines that protect against diphtheria toxin.
Diphtheria toxin (DT) is a protein exotoxin produced by the bacterium Corynebacteria diphtheria. The DT molecule is produced as a single polypeptide that is readily spliced to form two subunits linked by a disulfide bond, Fragment A (N-terminal xcx9cf21K) and Fragment B (C-terminal xcx9c37K), as a result of cleavage at residue 190, 192, or 193 (Moskaug, et al., Biol Chem 264:15709-15713, 1989; Collier et al., Biol Chem, 246:1496-1503, 1971). Fragment A is the catalytically active portion of DT. It is an NAD-dependent ADP-ribosyltransferase which specifically targets a protein synthesis factor termed elongation factor 2 (EF-2), thereby inactivating EF2 and shutting down protein synthesis in the cell. Fragment A consists of the diphtheria toxin C domain. Fragment A is linked to the diphtheria toxin Fragment B by a polypeptide loop. Fragment B of DT possesses a receptor-binding domain (the R domain) which recognizes and binds the toxin molecule to a particular receptor structure found on the surfaces of many types of mammalian cells. Once DT is bound to the cell via this receptor structure, the receptor/DT complex is taken up by the cell via receptor-mediated endocytosis. A second functional region on Fragment B (the T domain) acts to translocate DT across the membrane of the endocytic vesicle, releasing catalytically active Fragment A into the cytosol of the cell. A single molecule of Fragment A is sufficient to inactivate the protein synthesis machinery in a given cell.
Immunity to a bacterial toxin such as DT may be acquired naturally during the course of an infection, or artificially by injection of a detoxified form of the toxin (i.e., a toxoid) (Germanier, ed., Bacterial Vaccines, Academic Press, Orlando, Fla., 1984). Toxoids have traditionally been prepared by chemical modification of native toxins (e.g., with formalin or formaldehyde (Lingood et al., Brit. J. Exp. Path. 44:177, 1963)), rendering them nontoxic while retaining an antigenicity that protects the vaccinated animal against subsequent challenges by the natural toxin: an example of a chemically-inactivated DT is that described by Michel and Dirkx (Biochem. Biophys. Acta 491:286-295, 1977), in which Trp-153 of Fragment A is the modified residue.
Another method for producing toxoids is by the use of genetic techniques. Collier et al., U.S. Pat. No. 4,709,017 (herein incorporated by reference) disclosed a genetically engineered diphtheria toxin mutant that bears a deletion mutation at Glu-148 of diphtheria toxin. Glu-148 was originally identified as an active-site residue by photoaffinity labelling (Carroll et al., Proc. Natl. Acad. Sci. USA 81:3307, 1984; Carroll et al. Proc. Natl. Acad. Sci. USA 82:7237, 1985; Carroll et al., J. Biol. Chem. 262:8707, 1987). Substitution of Asp, Gln or Ser at this site diminishes enzymatic and cytotoxic activities by 2-3 orders of magnitude, showing that the spatial location and chemical nature of the Glu-148 side-chain greatly affects these activities (Carroll et al., J. Biol. Chem. 262:8707, 1987; Tweten et al., J. Biol. Chem. 260:10392, 1985; Douglas et al., J. Bacteriol. 169:4967, 1987). Similarly, Greenfield et al., U.S. Pat. No. 4,950,740 (herein incorporated by reference) disclosed genetically engineered mutant forms of DT in which the Glu-148 residue is deleted or replaced with Asn, and the Ala-158 residue is replaced with Gly. The DNA sequence and corresponding amino acid sequence of wild-type diphtheria toxin DNA are set forth in FIG. 1 (SEQ ID NOs:1 and 2, respectively).
The invention features diphtheria toxoids having multiple mutations as compared with wild-type diphtheria toxin. Thus, the invention features a polypeptide having a mutant diphtheria toxin C domain, a mutant diphtheria toxin T domain, and a mutant diphtheria toxin R domain, wherein the C domain has a mutation at Glu148, the T domain has a mutation at Glu349, and the R domain has a mutation at Lys 516 and/or Phe530 of wild-type diphtheria toxin. In various embodiments, the polypeptide includes any or all of the following mutations: Glu148Ser, Glu349Lys, Lys516Ala, and/or Phe530Ala.
The invention also features a polypeptide having a mutant diphtheria toxin C domain, a mutant T domain, and a mutant loop connecting the diphtheria toxin C and T domains, wherein the C domain has a mutation at Glu148, the T domain has a mutation at Glu349, and the loop has a mutation at Arg190, Arg192, and/or Arg193 of wild-type diphtheria toxin. In various embodiments, the polypeptide (or a mixture of polypeptides) includes any or all of the following mutations: Glu148Ser, Glu349Lys, Arg190Ser, Arg192Gly and/or Arg193Ser. In addition, all of the polypeptides of the invention bind sensitive cells with less affinity than does wild-type diphtheria toxin, and are capable of forming an immune complex with an antibody that specifically recognizes the R domain of wild-type diphtheria toxin.
These so-called xe2x80x9cmulti-mutantxe2x80x9d diphtheria toxoids of the invention can be used as vaccines to provide immunoprotection against diphtheria toxin and against infection by Corynebacteria diphtheriae. One approach to vaccination utilizes live, genetically engineered microorganisms (cells or viruses) expressing mutant toxin genes. The multi-mutant toxoids of the invention, and the DNAs which encode them, carry significantly less risk of reversion than do single residue deletion mutants, and so are good candidates for use in a live, genetically engineered vaccine cell that is capable of proliferating in the vaccinee. As discussed below, acellular vaccines also are within the invention.
The invention also includes vectors (e.g., plasmids, phages and viruses) including DNA sequences encoding the diphtheria toxoid mutants described herein. Expression of a diphtheria toxoid polypeptide of the invention can be under the control of a heterologous promoter, and/or the expressed amino acids can be linked to a signal sequence. A xe2x80x9cheterologous promoterxe2x80x9d is a promoter region that is not identical to the promoter region found in a naturally occurring diphtheria toxin gene. The promoter region is a segment of DNA 5xe2x80x2 to the transcription start site of a gene, to which RNA polymerase binds before initiating transcription of the gene. Nucleic acids encoding a diphtheria toxoid of the invention can be prepared as an essentially pure preparation, which is a preparation that is substantially free of other nucleic acid molecules with which a nucleic acid encoding diphtheria toxin is naturally associated in Corynebacterium. A nucleic acid encoding a diphtheria toxoid of the invention can be contained in a cell, or a homogeneous population of cells, preferably a B. subtilis, Bacillus Calmette-Guerin (BCG), Salmonella sp., Vibrio cholerae, Corynebacterium diphtheriae, Listeriae, Yersiniae, Streptococci, or E. coli cell. The cell is capable of expressing the diphtheria toxoid polypeptide of the invention.
Diphtheria toxoids that are xe2x80x9cimmunologically cross-reactivexe2x80x9d possess at least one antigenic determinant in common with naturally occurring diphtheria toxin, so that they are each bound by at least one antibody with specificity for naturally occurring diphtheria toxin. A diphtheria toxoid of the invention is immunologically cross-reactive with naturally occurring diphtheria toxin and possesses at least one of the mutations described herein.
The invention includes various vaccines that can be used to immunize a mammal (e.g., a human) against progression of the disease diphtheria, and against infection by the bacterium Corynebacterium diphtheriae. A vaccine of the invention can include any of the various DNAs encoding a diphtheria toxoid of the invention. Alternatively, a cell or virus expressing a nucleic acid of the invention, e.g., a live vaccine cell, can be used as a vaccine. Examples of suitable cells include B. subtilis, BCG, Salmonella sp., Vibrio cholerae, Listeriae, Yersiniae, Streptococci, Corynebacterium diphtheriae, and E. coli. A xe2x80x9clive vaccine cellxe2x80x9d can be a naturally avirulent live microorganism, or a live microorganism with low or attenuated virulence, that expresses an immunogen. A killed-cell vaccine can also be used.
One method for manufacturing a vaccine of the invention includes culturing a cell containing DNA encoding a diphtheria toxoid of the invention under conditions permitting proliferation of the cell and expression of the DNA, the cell being one that is suitable for introduction into an animal as a live-cell vaccine. The vaccine can be used in a method of immunizing a mammal against diphtheria by introducing an immunizing amount of a vaccine of the invention into the mammal.
In an alternative method of vaccination, an acellular vaccine that includes a nucleic acid encoding a diphtheria toxoid of the invention is introduced into the mammal. For example, a DNA vaccine can be administered by biolistic transfer, a method of delivery involving coating a microprojectile with DNA encoding an immunogen of interest, and injecting the coated microprojectile directly into cells of the recipient (Tang et al., Nature 356:152-154, 1992; hereby incorporated by reference). The diphtheria toxoid of the invention is then expressed from the DNA to stimulate an immune response in the recipient.
The polypeptides can be made by any of a variety of conventional methods, such as by culturing any of the various cells containing a DNA encoding a diphtheria toxoid of the invention under conditions permitting expression of the DNA. Included in the invention is an isolated mutant diphtheria toxin polypeptide, an xe2x80x9cisolatedxe2x80x9d polypeptide being one that is substantially free of cellular material, viral material, culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Generally, the polypeptide is a substantially pure preparation, meaning that at least 50% (by weight) (e.g., at least 75%, 90%, or 99%) of the protein present in the preparation is the diphtheria toxoid polypeptide of the invention.
A vaccine against diphtheria toxin can be formulated as a composition that includes a diphtheria toxoid polypeptide of the invention and an adjuvant. Examples of adjuvants include, but are not limited to, aluminum salts, bacterial endotoxins, Bacillus Calmette-Guerin (BCG), liposomes, microspheres (i.e., microencapsulation polymers used in orally administered vaccines), and Freund""s complete or incomplete adjuvant. An xe2x80x9cadjuvantxe2x80x9d is a substance that increases the immunogenicity of an antigen.
If desired, the diphtheria toxoid polypeptides of the invention can be covalently attached to a moiety, e.g., a polysaccharide or a second polypeptide. The moiety can serve as a carrier substance for the polypeptide or, alternatively, the diphtheria toxoid polypeptide of the invention can serve as a carrier substance for the moiety, preferably enhancing the immunogenicity of the moiety. A xe2x80x9ccarrier substancexe2x80x9d is a substance that confers stability on, aids, and/or enhances the transport or immunogenicity of an associated molecule.
A diphtheria toxoid of the invention can also be prepared as a fusion polypeptide that includes a diphtheria toxoid polypeptide covalently linked to a second polypeptide. The fusion polypeptide can be formulated as a vaccine, which can be used to immunize a mammal (e.g., a human patient) against diphtheria toxin or infection by Corynebacterium diphtheriae. The fusion polypeptide can be administered directly to the mammal in a method of immunization, or it can first be combined with an adjuvant. Alternatively, the DNA encoding the fusion polypeptide can be used directly as a vaccine, or it can be incorporated into a cell (e.g., a live vaccine cell) capable of expressing the fusion polypeptide, which cell can be used as a vaccine against diphtheria toxin. A xe2x80x9cfusion polypeptidexe2x80x9d is a polypeptide in which a diphtheria toxoid of the invention is linked to a second polypeptide sequence, typically by expression of a genetically engineered hybrid DNA.
The mutant diphtheria toxoids of the invention can be safely administered to a mammal in the form of an acellular polypeptide, a live attenuated vaccine strain that expresses the toxoid or a nucleic acid that expresses the toxoid in the vaccinee. The diphtheria toxoids of the invention are enzymatically dysfunctional and substantially free of any risk of reversion, even in a continuously proliferating microbial host.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.